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Abstract—Lignin acetate microspheres were synthesized in
uniform spherical shape through emulsion solvent evaporation
(ESE) technique. The objective of this work was to study the
influence of process variables on the mean particle size, size
distribution and morphology of lignin acetate microspheres.
There are many variables in ESE technique influencing the
properties of the microsphere, however, the focus of this study was
on four parameters; shear rate, mixing time, concentration of the
surfactant and the type of organic solvent. For synthesis of lignin
acetate microspheres, lignin was first modified to lignin acetate,
and then dissolved in the organic solvent. Then the organic phase
was added to the polyvinyl alcohol solution with a range of
different concentrations (0.0 – 2.0 w/v%). The mixture was
agitated at different shear rate with either homogenizer (high
shear mixer) for 5, 10, 20 and 30 seconds or magnetic stirrer (low
shear mixer) for 30 seconds. After the formation of the emulsion,
the liquid droplets of the organic phase were transformed into
solid microspheres by evaporation of the organic solvent. Size,
polydispersity index (PDI) and zeta-potential of the particles were
determined by Dynamic Light Scattering (DLS) technique.
Scanning Electron Microscopy (SEM) images were used to study
the morphology of the microspheres. The results showed that
uniform the microspheres were formed in about 1 µm at high
shear rate. We found that the size and morphology of the
microspheres depended on all four factors which were essential
factors in lignin acetate microspheres formation.
Keywords- Emulsion solvent evaporation technique, Lignin,
Microspheres, Preparation parameters.
I. INTRODUCTION
Lignin is produced in large quantities as by-product in the
pulp industries [1]. Major part of the lignin is used as
low-grade energy source for boilers, and only small portion
of lignin is utilized for value added products due to its
varying molecular weight, low functionality and unknown
molecular structure [2]. However, even with these
drawbacks, the interest for developing lignin-based products
is increasing due to the growing interest on sustainable
sources [3-5]. For instance, synthesis of lignin-based micro
particles were recently studied for their potential applications
in agricultural actives controlled release [6-9] drug controlled
release [9] coatings, cosmetics, auto, chemicals and
packaging industries [10].
Asrar and Ding (2006) [7] patented a method for producing
lignin-based matrix micro particles for the controlled release
of agricultural actives using emulsion solvent evaporation
(ESE) technique. The method produced lignin micro particles
in the range of 5.6–7.3 μm. However, there is no study
regarding the effect of preparation parameters on the
controlled synthesis of lignin micro particles in uniform
spherical shape. It is important to note that the synthesis of
uniform lignin micro particles in spherical shape requires an
appropriate method with controlled and precise preparation
parameters.
ESE technique is one of the most common techniques for
preparation of polymer microspheres [11]. There are two
different phases involved in this method; an organic phase
consisting of organic solvent and polymer, and aqueous
phase which included water and surfactant. Oil droplets
(dissolved polymer in an organic solvent) are formed in the
aqueous continuous phase when the organic phase is
intermixed with the aqueous phase [7]. After the formation of
the emulsion in the first step, the liquid droplets of the
organic phase are transformed into solid spherical particles
by removing the organic solvent from the emulsion [12, 13].
Accompanied by the solvent evaporation, the drops of the
dispersed phase become rich in polymer due to solvent
removal and they begin to solidify [14].
The organic solvent is chosen based on its ability to
dissolve the polymer, boiling point, miscibility with water,
and residual toxicity [15]. In ESE technique, the boiling point
of the organic solvent should be lower than the normal
boiling point of water. For instance, dichloromethane, ethyl
acetate, acetone, tetrahydrofuran are classified in this group
of solvents. However, the solubility of lignin is very low in
these organic solvents due to presence of hydrophilic moiety
in the lignin molecule [16]. The solubility characteristics of
the lignin macromolecules can drastically alter by removing
of hydroxyl groups due to altering their hydrophilic structure
and smaller amount H-bonds. Acetylation of lignin is a
technique usually done to improve the solubility of lignin in
organic solvents [17].
Although ESE technique is conceptually simple but many
variables can influence the final product [18]. Variables
influencing the final microsphere formation include: (i)
nature and solubility of polymer in organic solvent; (ii)
polymer concentration, composition and molecular weight;
(iii) organic solvent; (iv) concentration and nature of the
stabilizer/surfactant; (v) temperature; (vi) stirring/agitation
speed during emulsification process and; (vii) viscosity and
volume ratio of the dispersed and continuous phase [19].
However, the aim of this study was to define the effect of
only four parameters; shear rate, mixing time, concentration
of the surfactant and organic solvent on the size, size
distribution and the morphology of lignin acetate
microspheres.
Effect of Preparation Parameters on the
Formation of Lignin Acetate Microspheres Javad Sameni
1, Sally Krigstin
1, Mohini Sain
1,2
1 Center for Biocomposites and Biomaterials Processing, Faculty of Forestry, University of Toronto,
Toronto, ON, Canada. 2 Adjunct Professor, King Abdulaziz University (KAU), Jeddah, Saudi Arabia.
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II. EXPERIMENTAL
A. Materials
Commercial non-wood soda lignin was used in this study.
Solvents were purchased from commercial sources:
Dichloromethane (DCM) (Caledon, Georgetown, ON,
Canada), ethyl acetate (EA) (Caledon, Georgetown, ON,
Canada), acetone (ACE) (Caledon, Georgetown, ON,
Canada), tetrahydrofuran (THF) (Caledon, Georgetown, ON,
Canada), Pyridine (Caledon, Georgetown, ON, Canada),
polyvinyl alcohol (PVA) (Sigma, St. Louis, MO), acetic
anhydride (Caledon, Georgetown, ON, Canada). All the
chemicals used were analytical grade and used without
further purification.
B. Acetylation of lignin
1.0 g lignin was mixed with 40 ml of pyridine-acetic
anhydride (50-50%) solution. The mixture was allowed to
react at 90°C for 3 hrs in a sealed flask. The solids were
re-precipitated with 150 ml of hydrochloric acid solution (pH
= 1.0) and collected using a vacuum filtration technique. The
solids were washed with HCl solution and then with
deionized water. The collected solids were dried at 40°C and
stored in vials for further analysis.
C. Preparation of lignin acetate microspheres
10mg lignin acetate was dissolved in 1 mL organic solvent.
The organic phase was transferred into 10 mL aqueous phase
containing PVA with concentration of 0.0, 0.05, 0.1, 0.2, 0.5,
1 and 2 w/v%. The mixture was agitated with either magnetic
stirrer (800 rpm or 1000 rpm) for 30 seconds or homogenizer
(10,000-20,000 rpm) for 5, 10, 20 or 30 seconds. Then, the
emulsion was transferred to a beaker containing 50 mL of
water and stirred for 2-3 hrs with magnetic stirrer at room
temperature to allow the solvent to evaporate from the
mixture. The particles collected by centrifugation for 10 min
at 9000g, and washed with hot water for 2 times to remove
the surfactant. Samples were freeze-dried in and kept in
desiccator for further tests [7, 20]. Table 1 shows all the
preparation parameters for synthesis of lignin acetate
microspheres. The effect of each preparation parameter on
the particle size was analyzed by using one-way ANOVA
with significance when p<0.05.
D. Preparation of lignin acetate hollow spheres
At low shear rate (1000 rpm) a portion of particles (hollow
spheres) was remained on the surface of the water after
centrifugation due to their lower density than water. Particles
were collected from the surface of water, washed with
distilled water and dried in the freeze drier.
Determination of size and size distribution by using DLS
technique
The average diameter and size distribution of microspheres
were determined by using ZETASIZER NANO ZS Malvern
Instrument (Malvern, United Kingdom), at 25.0±0.1 °C. The
colloidal dispersions of lignin acetate microspheres were
prepared at 0.1% concentration of aqueous solution. The size
distribution graphs were obtained based on the relative
intensity of scattered light on the hydrodynamic diameter of
lignin microspheres. The relative intensity peaks were
normalized (the intensity of highest peak normalized to
unity) for all samples. At least three measurements were
carried out for each test.
Determination of zeta-potential by using DLS technique
The zeta-potential of lignin acetate microspheres were
determined using ZETASIZER NANO ZS Malvern
Instrument (Malvern, United Kingdom), at 25.0±0.1 °C. A
colloidal dispersion of lignin microspheres was prepared in
an aqueous solution with concentration of 0.01%. At least
three measurements were carried out for each test.
Morphology of the particles by Scanning Electron
Microscopy
The morphology of microspheres was observed under
scanning electron microscopy (SEM). SEM was used to
characterize the morphology of the lignin particles. Samples
were covered by a fine gold layer (10 nm) and observed on a
JEOL field emission microscope (5 kV). Diameters of the
lignin particles were measured by using ImageJ software.
The mean diameter of each sample was calculated based on
the measurements of 100 randomly selected particles.
Colloidal stability test
Stability of the lignin acetate microspheres suspensions
were studied by analyzing the particles size over time. Lignin
acetate microspheres were immersed in water at room
temperature for 60 days. Particles size was measured by
using DLS in 15, 25, 35 and 60 days to evaluate the
occurrence of aggregation due to cluster formation [21]. The
results were reported as mean (±SD). Statistical analysis were
performed by using one-way analysis of variance (ANOVA)
with significance (p<0.05). The morphology of the particles
on the 60th day was studied by analyzing SEM images.
Sample Mixer
Shear
rate
(rpm)
Mixing
Time
(Sec)
PVA
Conc.
(w/v %)
1
Magnetic
stirrer 1000 30 0
2
Magnetic
stirrer 800 30 0.2
3
Magnetic
stirrer 1000 30 0.2
4 Homogenizer 10000 5 0.2
5 Homogenizer 10000 10 0.2
6 Homogenizer 10000 20 0.2
7 Homogenizer 10000 30 2
8 Homogenizer 10000 30 1
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9 Homogenizer 10000 30 0.5
10 Homogenizer 10000 30 0.2
11 Homogenizer 10000 30 0.1
12 Homogenizer 10000 30 0.05
13 Homogenizer 10000 30 0
14 Homogenizer 12500 30 0.2
15 Homogenizer 15000 30 0.2
16 Homogenizer 20000 30 0.2
TABLE 1 Preparation parameters for synthesis of lignin acetate microspheres
III. RESULTS AND DISCUSSION
A. The influence of shear rate
Generally the size of droplets in an emulsion is inversely
related to the magnitude of shear stresses. Therefore, smaller
microspheres are formed by increasing the shear rate. It is
reported that increasing stirring speed produced smaller
particles using ESE technique [22]. As the speed of the motor
or the power of the sonicator is increased, the size of the
dispersed droplets decreases [11]. Therefore, if high shear is
produced by homogenizer or sonicator, the droplets become
much smaller than the droplets in the emulsion produced by
conventional agitation. This phenomenon strongly supports
the concept that the stronger shear forces and increased
turbulences that are generated at high stirring speed could
breakdown the droplets into smaller sizes [23].
Figure 1a shows the particle size distribution of lignin
acetate microspheres which prepared at low shear (800 rpm
and 1000 rpm) and high shear rate (10,000 rpm). The low
shear was applied by magnetic stirrer and high shear by
homogenizer. The size of the particles prepared by using
magnetic stirrer was determined by imageJ (Table 2).
Particles were formed in larger size and in wider distribution
by using magnetic stirrer. The average particle size at 800
rpm and 1000 rpm was 13.6 µm and 10.6 µm, respectively.
Figure 1b shows the size distribution of lignin acetate
microspheres which prepared by using homogenizer at
different shear rates (10,000-20,000 rpm). All samples
showed unimodal size distribution. However, it seems that
the distributions of lignin acetate microspheres were slightly
shifted to lower sizes at higher shear rate.
Fig 1. Particle size distribution of lignin acetate microspheres at
different shear rate; Particle size was determined by a) imageJ
software and b) DLS technique
Table 2 minimum, maximum and mean particle size prepared
by using magnetic stirrer and measure by imageJ software
Shear rate (rpm) Particle size (µm)
Min. Max. Mean
800 3.8 25.0 13.6
1,000 2.0 18.0 10.6
10,000 0.5 2.4 1.0
Figure 2 shows the effect of high shear rate on the average
particle size and PDI of the lignin acetate microspheres. It is
clear that the average size of microspheres was decreased
from 1075 nm to 886 nm when the shear rate was increased
from 10,000 rpm to 20,000 rpm. Significance of the influence
was statistically confirmed by one-way ANOVA (p < 0.05).
Although, the size distribution became slightly narrower by
increasing the shear rate, but the value of PDI was increased
at higher shear rates. It has been reported that the particle size
distribution of poly(lactic-glycolic) acid (PLGA) and
Eudragit RS microspheres decreased when stirring speed
increased [18, 24]. SEM images (Fig. 3) shows the obvious
differences between the effects of the high and low shear rate
on the particle size.
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Fig 2. Average particle size (Z-Ave) and polydispersity index (PDI) of lignin acetate microspheres at different shear rate applied by
homogenizer
Fig 3. SEM images of lignin acetate microspheres which prepared by using magnetic stirrer at low shear (800 rpm and 1000 rpm)
and homogenizer at high shear rate (10,000-20,000 rpm).
10000 rpm 12500 rpm
15000 rpm 20000 rpm
800 rpm 1000 rpm
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Many other factors related to agitation have also an
influence on the size of the microspheres, such as the
geometry of the reactor, the number of impellers and the
impeller’s diameter [25]. Based on the Kolmogoroff/Hinze
model [26] the correlation between the agitation rate and the
diameter of the agitator could be expressed as:
Eq. 1
Where;
dmax = the largest drop size which can be formed under
turbulence (m)
D = the diameter of the agitator (m)
ρc = the density of continuous phase (kg/m3)
N = the agitation rate (turns/s)
σ= the interfacial tension between the continuous phase and
the dispersed phase (N/m)
c1 = a constant value which depends on the factors linked to
the agitation conditions.
If we assume a constant density for the continues phase and a
constant interfacial tension between the continuous phase and
the dispersed phase, then;
Eq. 2
where C2 is a constant.
From Eq. (1) and Eq. (2), it is clear that the maximum size
of microspheres is decreased by increasing the agitation rate
[18, 24, 27]. Figure 4 shows the relationship between the
largest particle size of lignin acetate microspheres with the
diameter of the agitator and the agitation rate.
Fig 4 the relationship between the diameter of the agitator (D)
and the agitation rate (N) with the maximum size of the lignin
acetate microspheres (dmax)
Formation of lignin acetate hollow spheres
Figure 5 shows SEM images of the lignin acetate hollow
spheres. The average diameter and the thickness of the lignin
acetate hollow spheres were determined by imageJ software
on the SEM images. It was found that the thickness of the
hollow spheres was about 1-3 µm, and the average particle
size was about 58 µm (Fig. 6).
Polymer hollow spheres have shown the potential in
variety applications ranging from controlled release to
catalysis [28, 29]. Their ability to compartmentalize a large
quantity of chemical actives and their large surface area make
these particles particularly useful [30]. For instance, Liu et al
(2014) [31] reported the fabrication of PLGA hollow
microcapsules through double emulsion method having a size
of 2.5 μm and loaded with an anticancer drug for targeted
drug delivery to cancer cells. In another study, hollow
structure of poly(3-hydroxybutyrate-co-3-hydroxyvalerate)
was synthesized through ESE technique for the sustained
release of anti-cancer drug [32].
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Fig 5 SEM images of lignin acetate hollow spheres
Fig 6 Particle size distribution of lignin acetate hollow sphere
B. The influence of surfactant concentration
Figure 8 shows the particle size distribution of lignin acetate
microspheres prepared at different PVA concentration (0.05 -
2.0% w/v). Unimodal distribution was observed for all
samples. The size distribution was shifted to smaller particles
by increasing the PVA concentration from 0.05% to 1%.
However, by increasing the PVA from 1% to 2%, the size
distribution was moved to greater particles.
Surfactants play an important role in the formulation of
microspheres and in their resulting shape and size. The main
role of the surfactant is to prevent the emulsion droplets from
coalescing [23]. The surfactant molecules are located in the
interface between the aqueous phase and organic phase. The
concentration and properties of the surfactants will affect the
total surface area of the particles and may change the final
particle size [33].
Fig 7 particle size distributions of lignin acetate microspheres at
different PVA concentration
Figure 8 shows that the average particle size of lignin
acetate microspheres was decreased by addition of PVA from
0.05% to 1%, and then increased by addition of 2% PVA.
Therefore, the smallest particle size (744 nm) was obtained
by using 1% PVA in the formulation. However, the
polydispersity of particles at 1% PVA was the highest
(PDI=0.22) in comparison with other samples. According to
ANOVA, the PVA concentration is found to have a
significant influence on the lignin acetate particle size
(p<0.05).
Silva et al., [20] reported that the PVA concentrations
below 1% led to a larger particle size of PLGA microspheres,
because smaller particles have a higher total interfacial area
compared to the large particles, thus they require a higher
concentration of the surfactant. Therefore, the addition of
higher surfactant concentration to the solution results in
decreased particles size. In another study, an increase in size
of PLGA nanoparticles at high PVA concentration have been
reported [34]. These contradictory findings were clarified by
Budhian [35] who proposed two competing effects at high
PVA concentration. The size of the particles decreases due to
enhanced interfacial stabilization while the size of the
particles increases due to increased viscosity of the aqueous
phase.
Fig 8 Average particle size and PDI of lignin acetate
microspheres at different PVA concentration
80 µm
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SEM images show the effect of surfactant concentration on
the morphology of lignin acetate microspheres (Fig. 10). A
comparison between SEM images shows that the formation
of lignin acetate particles with surfactant was completely in
spherical shape and had a smooth surface. Absence of
surfactant causes particle shrinkage and rough surface on the
particles.
Fig 9 SEM images of lignin acetate microspheres at different PVA concentration (0.0-2.0%). Agitation rate was 10,000 rpm for all
cases, unless stated on the image.
0.2% 0.5%
1% 2%
0.05 % 0.1%
0% 0% (1,000 rpm)
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C. The influence of mixing time
Figure 10 presents size distribution of lignin acetate
microspheres that were prepared at different mixing times (5,
10, 20 and 30 sec.) by using homogenizer and constant PVA
concentration (0.2%w/v). It shows uniform size distribution
for all samples, but obvious differences of particle sizes was
observed between samples. Larger particles were formed
during shorter mixing time.
Fig 10. Particle size distribution of lignin acetate microspheres
at different mixing time
Figure 11 shows the average particle size and PDI of lignin
acetate microspheres at different mixing time. The average
particle size was 1767 nm, 1291 nm, 1062 nm and 1075 nm at
5, 10, 20 and 30 second of mixing time, respectively.
ANOVA showed significant difference between lignin
acetate particle size at different mixing times (p<0.05). PDI
was decreased from 0.216 to 0.110 when the mixing time
increased from 5 sec to 30 sec.
Short mixing time yields coarse particles due to less
magnitude of shear stress applied, while at longer time, the
increase in the energy density directly by increasing the shear
stresses and results in more efficient droplet breakdown [35].
Therefore, increasing the mixing time decreases the particles
mean size due to reduction of emulsion droplets through
sufficient shear forces. Figure 13 shows SEM images of
lignin acetate microspheres at different mixing time.
Fig 11 Average particle size and PDI of lignin acetate
microspheres at different mixing time
Fig 12 SEM images of lignin acetate microspheres at different mixing time
D. The influence of organic solvent
5 Sec. 10 Sec.
20 Sec. 30 Sec.
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SEM images show the formation of lignin acetate
microsphere when DCM, ACE, EA and THF were chosen as
organic solvents (Figure 14). Homogenizer was used as
agitator, mixing time was fixed at 30 Sec. and PVA
concentration was 0.2 w/v%. Although lignin acetate was
completely soluble in all four organic solvents but lignin
acetate microspheres were formed only in DCM and EA.
Fig 13 SEM images of lignin acetate microspheres at different organic solvents
Figure 14 illustrates the size distribution of the lignin acetate
microspheres. A comparison between the size distributions
indicates a wider size distribution when EA compared with
DCM.
Fig 14. Particle size distribution of lignin acetate microspheres
with different organic solvents
Table 3 shows the average size, PDI, and zeta-potential of
the lignin acetate microspheres when the organic solvent was
EA and DCM. The average size of the particles was 1881 nm
and 1075 nm for EA and DCM, respectively. The PDI was
lower (narrower distribution) for DCM (0.118) in
comparison with EA (0.173). The results show that the
zeta-potential of the lignin acetate microspheres with EA is
-45.5 mV while with DCM is -36.7 mV. The value of
zeta-potential depends on the chemicals involved in the
synthesis process [36]. The negative zeta-potential is caused
by the residue of the PVA surfactant on the particles surface
[37], residue of the solvent and surface free carboxylic acid
groups on the lignin acetate. The larger zeta potential values
of both samples expected to exhibit high colloidal stability
[21].
Table 3. Average size, PDI and zeta-potential of lignin acetate microspheres in EA and DCM
Acetone
Ethyl acetate
Tetrahydrofuran
Dichloromethane
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Organic solvent Z-Ave (d.nm) PDI zeta -potential (mV)
Ethyl acetate 1881(±46.5) 0.173 (±0.030) -45.5 (±2.13)
Dichloromethane 1075 (±6.9) 0.118 (±0.018) -36.7 (±0.46)
It is clear that formation of particles was mainly depended
on the physical properties of the organic solvent. Physical
properties of the selected organic solvents are presented in
Table4. ACE and THF are diffused rapidly from the
dispersed phase into aqueous phase due to their miscibility in
water. Therefore, lignin acetate was dispersed in aqueous
phase in irregular shapes before it was formed and stabilized
by the surfactants molecules. On the other hand, DCM is
immiscible in water and the solubility of EA is low in water
(8.3 g/100mL). Therefore, DCM and EA remained in the
emulsion droplets for a while before diffusing into the
aqueous phase. The results indicate that lignin acetate
particles are formed and solidified in uniform size and shape
by using DCM and EA in the process. Interfacial tension of
DCM is higher than EA, and it is immiscible in water.
Therefore, DCM resulted in successful formation of smaller
lignin acetate microspheres with narrow size distribution in
comparison with EA. The higher solubility of EA in water
may results in significant aggregation leading to larger
particles.
Dichloromethane (chlorinated solvents that challenge
human safety and environmental concern) have been widely
used as a good organic solvent in ESE technique. In order to
reduce the use of these toxic solvents, many attempts have
been made to prepare the polymer microspheres using a
solvent with lower toxicity, such as ethyl acetate, as the
dispersing solvent. The effect of ethyl acetate as a dispersing
solvent was also studied in the production of different
polymers such as PLGA microspheres [15].
Table 4 Some physical properties of selected organic solvent [36, 38]
Solvent Bp (ºC) Density (g/cm3)
at 20 ºC
Solubility in water (wt%) at
20-25ºC
Viscosity
(Cp)
Interfacial tension
(dyne/cm)
DCM 39.8 1.3255 1.32 0.44 28.3
EA 76.7 0.9018 8.7 0.46 1.3
ACE 56.0 0.7910 Miscible 0.32 -
THF 66.0 0.8892 Miscible 0.48 -
E. Colloidal stability of the lignin acetate microspheres
Table 5 shows the particle size of the microspheres on the
first day of preparation and after 15, 25, 35 and 60 days in a
neutral suspension at room temperature. According to
ANOVA analysis, the difference between the particle size
was insignificant up to 35 days (p>0.05) when DCM used in
the formulation, while the particle size was slightly increased
at 60 days (p<0.05). ANOVA results also showed that the
mixture of lignin acetate microspheres prepared with EA
were stable in the first 15 days of the stability test (p>0.05),
but it was significantly increased on the 60th
day of the test.
The reason that the suspension of lignin acetate microspheres
were remained in stable condition for several days is due to
their high negative zeta potential.
Table 5 Average size of lignin acetate microspheres samples subjected to stability test at room temperature over time. Mean value
(±Standard Deviation)
Time (Days) DCM EA
1 1075 (±6.9) 1881(±46.5)
15 1078 (±10.0) 1957 (±10.0)
25 1085 (±5.3) 2004 (±14.5)
35 1094 (±16.3) 2117 (±50.5)
60 1137 (±21.5) 2184 (±22.1)
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SEM images of the lignin acetate microspheres in DCM
and EA show that the particles remained in stable shape
without shrinking and collapse on 60th
day (Figure 16).
Fig 15 Lignin acetate microspheres after 60 days in neutral suspension
IV.CONCLUSION
Lignin acetate microspheres were prepared successfully
using the ESE technique. Lignin acetate microspheres were
formed with a size of 1075 nm and uniform distribution
(PDI=0.118) by using homogenizer (high shear rate at
10,000rpm), sufficient time for mixing (30 seconds) and
using at least 0.2%w/v of PVA. It is clear that the properties
of lignin acetate microspheres were affected by altering
preparation parameters. The results showed that the particle
size, size distribution, morphology and zeta-potential of
lignin acetate microspheres depends on the shear rate, mixing
time, surfactant concentration and organic solvent which are
essential factors in lignin acetate microsphere formation.
Lignin acetate microspheres were formed with slightly larger
size (1881 nm), wider size distribution (PDI=0.173) and
higher negative charge (-45.5 mV) when EA used in the
formulation. DLS results and ANOVA analysis showed that
the suspension of lignin acetate microspheres which prepared
by using DCM were in stable condition for at least 35 days,
while the suspension prepared by using EA was stable for at
least 15 days.
ACKNOWLEDGEMENT
The authors are grateful for the financial support from
ORF-RE funding programs.
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